U.S. patent number 4,436,657 [Application Number 06/519,725] was granted by the patent office on 1984-03-13 for antitumor glycoprotein substance and preparation thereof.
This patent grant is currently assigned to Maruzen Oil Co., Ltd.. Invention is credited to Kiyoshi Minami, Kazuya Nakamichi, Takuma Sasaki, Yakudo Tachibana.
United States Patent |
4,436,657 |
Sasaki , et al. |
March 13, 1984 |
Antitumor glycoprotein substance and preparation thereof
Abstract
A novel glycoprotein substance possessing a high antitumor
activity is provided, which is recovered and isolated from the
liquid portion coming from cooking of raw scallop with a hot
aqueous solvent or vapor of such solvent and which is amphoteric
electrolyte in nature with an average molecular weight of about
470,000 and with an isoelectric point of pH 5.5.
Inventors: |
Sasaki; Takuma (Tokyo,
JP), Nakamichi; Kazuya (Sohka, JP),
Tachibana; Yakudo (Sohka, JP), Minami; Kiyoshi
(Koshigaya, JP) |
Assignee: |
Maruzen Oil Co., Ltd. (Osaka,
JP)
|
Family
ID: |
15193358 |
Appl.
No.: |
06/519,725 |
Filed: |
August 2, 1983 |
Foreign Application Priority Data
|
|
|
|
|
Aug 9, 1982 [JP] |
|
|
57-137210 |
|
Current U.S.
Class: |
530/395; 530/412;
530/857; 424/547; 530/416 |
Current CPC
Class: |
A61P
35/00 (20180101); A61K 35/60 (20130101); A23J
1/04 (20130101); Y10S 530/857 (20130101) |
Current International
Class: |
A61K
35/60 (20060101); A23J 1/04 (20060101); A23J
1/00 (20060101); A61K 35/56 (20060101); A23J
001/04 (); C07G 007/00 () |
Field of
Search: |
;260/112R |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
J of National Cancer Institute, vol. 60, No. 6, pp. 1499-1500, Jun.
1978, Sasaki et al..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Larson and Taylor
Claims
What we claim is:
1. A glycoprotein substance possessing an antitumor activity which
is recovered from the liquid portion which comes from cooking of
raw scallop with a hot aqueous solvent or with the vapor of such
solvent and which has the following characteristics:-
(1) Appearance: White, powdery solid.
(2) Solubility: Soluble easily in water, but insoluble in organic
solvents such as methanol, ethanol and acetone.
(3) Acidic or basic nature: Amphoteric electrolyte having an
isoelectric point of pH 5.5.
(4) Infrared absorption spectrum (pelleted in KBr): With
characteristic absorption peaks at 3500.about.3300, 1660 and 1550
cm.sup.-1.
(5) Ultraviolet absorption spectrum in an aqueous solution: With a
characteristic absorption peak .lambda..sub.max H.sub.2 O at 255
nm.
(6) Color reactions: Positive in biuret reaction, xanthoproteic
reaction, phenolic reagent reaction according to Lowry-Folin
method, anthrone-sulfuric acid reaction, phenol-sulfuric acid
reaction and cysteine-sulfuric acid reaction.
(7) Average molecular weight: About 470,000 as measured by
gel-electrophoresis.
2. A glycoprotein substance possessing an antitumor activity which
is recovered from the liquid portion coming from cooking of raw
scallop with a hot aqueous solvent or with the vapor of such
solvent by concentrating the liquid portion to a smaller volume or
to dryness to give a concentrate or dry powder, dissolving the
concentrate or dry powder in water or an aqueous solvent to form an
aqueous solution thereof and subjecting the aqueous solution to a
series of treatments comprising ion-exchange chromatography with a
basic anion exchanger and molecular weight-fractionating treatments
comprising gel-filtration and electrophoresis, in any desired
sequence, and which has the following characteristics:-
(1) Appearance: White, powdery solid.
(2) Solubility: Soluble easily in water, but insoluble in organic
solvents such as methanol, ethanol and acetone.
(3) Acidic or basic nature: Amphoteric electrolyte having an
isoelectric point of pH 5.5.
(4) Infrared absorption spectrum (pelleted in KBr): With
characteristic absorption peaks at 3500.about.3300, 1660 and 1550
cm.sup.-1.
(5) Ultraviolet absorption spectrum in an aqueous solution: With a
characteristic absorption peak .lambda..sub.max H.sub.2 O at 255
nm.
(6) Color reactions: Positive in biuret reaction, xanthoproteic
reaction, phenolic reagent reaction according to Lowry-Folin
method, anthrone-sulfuric acid reaction, phenol-sulfuric acid
reaction and cysteinesulfuric acid reaction.
(7) Average molecular weight: About 470,000 as measured by
gel-electrophoresis.
3. A glycoprotein substance according to claim 2 wherein the
treatments of the aqueous solution for the isolation of the desired
glycoprotein substance therefrom are carried out in a buffer
solution having a strong buffering action.
4. A process for thc preparation of a water-soluble, antitumor
glycoprotein substance having an average molecular weight of about
470,000 and an isoelectric point of pH 5.5 from scallop which
comprises recovering the liquid portion which comes from cooking of
raw scallop with a hot aqueous solvent or with vapor of such
solvent and which is to be discarded as waste, concentrating the
liquid portion thus recovered to a smaller volume or to dryness to
yield a concentrate or dry powder, dissolving the concentrate or
dry powder in water or an aqueous solvent to form an aqueous
solution thereof and subjecting the aqueous solution to a series of
treatments comprising ion exchange chromatography with a basic
anion exchanger and molecular weight-fractionating treatments
comprising gel-filtration and electrophoresis, in any desired
sequence, whereby isolating a fraction consisting essentially of a
glycoprotein substance which is adsorbable on a basic anion
exchanger and which has an average molecular weight of about
470,000 and an isoelectric point of pH 5.5.
5. A process according to claim 4 wherein after the concentrate or
dry powder is dissolved in water, the aqueous solution so formed is
subjected to ion-exchange chromatography with a basic anion
exchanger to recover a fraction adsorbed on the basic anion
exchanger, and the fraction is then subjected to molecular
weight-fractionating treatments comprising gel filtration and
electrophoresis to isolate a fraction consisting essentially of a
glycoprotein substance having an average molecular weight of about
470,000 and an isoelectric point of pH 5.5.
6. A process according to claim 4 wherein the treatments of the
aqueous solution for the isolation of the desired glycoprotein
substance therefrom are carried out in a buffer solution having a
strong buffering action.
Description
FIELD OF THE INVENTION
This invention relates to a novel antitumor glycoprotein substance
and a process for the preparation thereof from scallop.
BACKGROUND OF THE INVENTION
We have already found and reported that several antitumor
substances are obtained from meat portion of shellfish and exhibit
wide antitumor spectra with little or no cytotoxicity (refer to
Japanese Patent Publication No. 8088/82 and Japanese Patent KOKAI
Nos. 41314/79 and 41315/79). To be concrete, the first substance we
found is a water-soluble, thermally stable glycoprotein having a
molecular weight range within the limits of 100,000 and 300,000
(Japanese Patent Publication No. 8088/82). Japanese Patent KOKAI
No. 41314/79 describes four substances all of which are
water-soluble glycoprotein substances having an average molecular
weight of about 20,000 and an isoelectric point of pH 4.5 with
somewhat different physical properties from one another and are
extracted from meat portion of scallop from which the liver has
been removed. Japanese Patent KOKAI No. 41315/79 provides a
water-soluble glycoprotein substance having an average molecular
weight of 10,000.about.30,000 which is obtained from meat portion
of shellfish, particularly of scallop, wreath shell, tokobushi
(Haliotis japonica) and pearl-oyster, from which the liver has been
removed.
After that, we have further found that the liquid portion which
comes from cooking of raw shellfish with a hot aqueous solvent or
with the vapor of such solvent for taking up edible portions
thereof and which is to be discarded as waste can also serve as raw
material from which water-soluble, macromolecular glycoprotein
substances similar to those already obtained from shellfish as
above-mentioned are recovered and that these substances have a
range of molecular weights within the limits of from 10,000 to
300,000 and possess a significant antitumor activity (refer to T.
Sasaki et al, U.S. patent application Ser. No. 404,971, now U.S.
Pat. No. 4,390,468).
Thus, the known antitumor substances derived from shellfish may be
divided into two broad classes, namely water-soluble glycoproteins
having a molecular weight range within the limits of 10,000 and
30,000, typically of around 20,000, and those having a molecular
weight range within the limits of 100,000 and 300,000.
We have continued our investigations on antitumor substances
derived from scallop, particularly from the liquid waste portion
coming from cooking of raw scallop and now found the presence in
the dry powder recovered from said waste liquid of new antitumor
substance having much higher average molecular weight than those of
all the known antitumor glycoprotein substances derived from
shellfish. On the basis of this discovery, we have followed up our
study thereon and successfully isolated a new antitumor
glycoprotein substance and determined physico-chemical properties
and antitumor activities thereof.
SUMMARY OF THE INVENTION
Accordingly, it is the main object of this invention to provide a
novel glycoprotein substance possessing a significant antitumor
activity. Another object of this invention is to provide a process
for the preparation of the novel glycoprotein substance from the
liquid portion, i.e. waste liquor, coming from cooking of raw
scallop. A further object of this invention is to provide an
antitumor agent with a low toxicity. These and other objects of
this invention will become clear from the following
descriptions.
According to the first aspect of this invention, therefore, there
is provided a glycoprotein substance possessing an antitumor
activity which is recovered from the liquid portion which comes
from cooking of raw scallop with a hot aqueous solvent or with the
vapor of such solvent and which has the following
characteristics:
(1) Appearance: White, powdery solid.
(2) Solubility: Soluble easily in water, but insoluble in organic
solvents such as methanol, ethanol and acetone.
(3) Acidic or basic nature: Amphoteric electrolyte having an
isoelectric point of pH 5.5.
(4) Infrared absorption spectrum (pelleted in KBr): With
characteristic absorption peaks at 3500.about.3300, 1660 and 1550
cm.sup.-1.
(5) Ultraviolet absorption spectrum in an aqueous solution: With a
characteristic absorption peak .lambda..sub.max.sup.H.sbsp.2.sup.O
at 255 nm.
(6) Color reactions: Positive in biuret reaction, xanthoproteic
reaction, phenolic reagent reaction according to Lowry-Folin
method, anthrone-sulfuric acid reaction, phenol-sulfuric acid
reaction and cysteine-sulfuric acid reaction.
(7) Average molecular weight: About 470,000 as measured by
gel-electrophoresis.
BRIEF DESCRIPTION OF THE DRAWINGS
In the accompanying drawings:
FIG. 1 shows an elution pattern depicted in gel-filtration
chromatography carried out in a step for isolating a water-soluble
glycoprotein substance according to this invention, more concretely
an elution pattern depicted in gel-filtration chromatography of an
intermediate substance separated by DEAE ion-exchanging
chromatography in Example 2 hereinafter given, wherein the abscissa
axis represents fraction numbers of the eluate, the ordinate axis
represents absorbance shown as optical density, the white circles
(S) show absorbance at wave length of 490 nm of each eluate
fraction colored by the method of Dubois et al. (see J. Anal.
Chem., 28, 350-356 (1956)) as an indicator of the carbohydrate
content of fractions and the black circles (P) show absorbance at
wave length of 750 nm of each eluate fraction colored by Lowry
method as an indicator of the protein content of fractions.
FIG. 2 shows infrared absorption spectrum of a sample of
glycoprotein substance according to this invention (Sample E
obtained in Example 2 hereinafter given) pelleted in KBr and
FIG. 3 shows ultraviolet absorption spectrum of the same sample in
the form of a 0.1 mg/ml aqueous solution.
DETAILED DESCRIPTION OF THE INVENTION
The novel glycoprotein substance according to this invention is
derived from natural sources, particularly from the liquid portion
coming from the cooking of raw scallop in an aqueous solvent or
with vapor of such solvent, and is a homogeneous substance of high
average molecular weight which shows a single peak both in liquid
chromatography and in gelfiltration chromatography and shows a
single band in electrophoresis such as disc electrophoresis, which
corresponds to Sample E obtained in Example 2 hereinafter given.
This substance has the following physico-chemical properties:
(1) Elementary analysis: C 40.2; H 6.4; N 7.1; S 0.1; P 0.1; Ash
1.0% by weight.
(2) Molecular weight: About 470,000 as average molecular weight
obtained by SDS-5% polyacrylamide gel-electrophoresis wherein
comparison is made between Rf value (relative mobility) of the
sample and those values of standard substances to estimate the
molecular weight of the former correlatively.
(3) Rf value (relative mobility) in electrophoresis: A single peak
band at Rf value of 0.04 relative to that of Bromophenol blue as
standard in electrophoresis on 7.5% acrylamide gel in a buffer
solution at pH 8.0.
(4) Melting point: No definite melting point and no definite
decomposition point are seen, but some decomposition is observed at
240.degree. C.
(5) Ultraviolet absorption spectrum: An aqueous solution of the
sample reveals the UV absorption spectrum as shown in FIG. 3 with a
characteristic absorption peak
.lambda..sub.max.sup.H.sbsp.2.sup.2.sup.O at 255 nm.
(6) Infrared absorption spectrum (pelleted in KBr): As shown in
FIG. 2 with characteristic absorption peaks at 3500.about.3300,
2960, 2940, 1660, 1550 and 1400 cm.sup.-1.
(7) Color reactions: Positive in biuret reaction, xanthoproteic
reaction, phenolic reagent reaction according to Lowry-Folin
method, anthrone-sulfuric acid reaction, phenol-sulfuric acid
reaction and cysteine-sulfuric acid reaction.
(8) Acidic or basic nature: Amphoteric electrolyte having an
isoelectric point of pH 5.5.
(9) Solubility: Soluble in water, but insoluble in organic solvents
such as methanol, ethanol and acetone.
(10) Appearance: White, powdery solid.
(11) Carbohydrate content: 29.4% by weight calculated in terms of
glucose when measured by the phenol-sulfuric acid method.
(12) Amino acids in hydrolyzate: When hydrolyzed in 6 N HCl at
105.degree..about.110.degree. C. for 24 hours, the resulting
hydrolyzate contains at least the following amino acids: aspartic
acid, hydroxyproline, threonine, serine, glutamic acid, proline,
glycine, alanine, cysteine, valine, methionine, isoleucine,
leucine, tyrosine, phenylalanine, histidine, 3-methylhistidine,
hydroxylysine, lysine, arginin.
(13) Carbohydrates in hydrolyzate: When hydrolyzed in 1N HCl at
80.degree. C. for 3 hours, the hydrolyzate shows by gas
chromatography to contain at least the following neutral
carbohydrates: fructose, galactose, fucose, mannose.
Judging from the above-mentioned properties of the sample
substance, it is believed that the substance according to this
invention consists essentially of a water-soluble, high molecular
weight glycoprotein.
As above-mentioned, the substance according to this invention has a
high average molecular weight of about 470,000 and a relatively low
isoelectric point of pH 5.5. On the other hand, the concentrate or
dry powder derived from the waste liquid portion coming from
cooking of raw scallop contains a variety of amphoteric
electrolytes having different isoelectric points, thus exhibiting a
strong pH-buffering action. When dissolved in distilled water, the
concentrate or dry powder shows a pH of about 5.5. When dissolved
in 0.1 M phosphate buffer solutions having different pH values
lower than 7.0, the concentrate or dry powder shows a pH of
5.2.about.6.0 with a tendency of lowering the pH value with the
increase in the amount of concentrate or dry powder dissolved.
Possibly owing to these behaviors of the concentrate or dry powder,
substances having an isoelectric point of pH of around 5.5 such as
the substance according to this invention could not be eluted or
dissolved in an eluent or solvent used during various fractionation
and isolation operations for the purpose of recovering
water-soluble glycoprotein substances possessing useful antitumor
activities from the concentrate or dry powder because those
substances would form insoluble precipitate or adsorb on various
gels used during such operations. This is, we think, the reason why
the substance according to this invention could not have been
detected until now.
As explained hereinbefore, this invention is based on the discovery
during our investigations on antitumor substances contained in the
concentrate or dry powder in question that fractions corresponding
to such high molecular weight substances as about
450,000.about.500,000 exhibit a very high antitumor activity. This
is quite unknown yet, thus unexpected in the art.
As a result of our careful examination of all the above-mentioned
properties of the substance of this invention in comparison with
those properties of known antitumor substances derived from
shellfish given in literature including typically Japanese Patent
Publication No. 8088/82, Japanese Patent KOKAI Nos. 41314/79 and
41315/79, U.S. patent application Ser. No. 404,971 and Journal of
National Cancer Institute, 60, 6, 1499-1500 (1978), we believe that
the substance according to this invention is not identical with any
of those disclosed in literature but novel and useful as antitumor
agent.
The glycoprotein substance according to this invention can be
derived from scallop. To be concrete, this substance may be
prepared starting from the liquid portion to be discarded as waste
which comes from cooking of raw scallop with a hot aqueous solvent
or with the vapor of such solvent. The liquid portion which
includes a condensate obtained in the cooking of raw scallop may be
used as it is or in a concentrate or a dry powder derived
therefrom. The isolation of the substance from the said liquid
portion and the purification of the substance thus isolated may be
effected in a manner known per se, that is by applying any one of
or any combination of two or more of known means usually used for
the isolation and purification of protein and carbohydrate
substances in the art, such as, for example, centrifugation,
dialysis, ultrafiltration, gel-filtration chromatography,
ion-exchange chromatography, chromatofocusing, ion exchange
resin-treatment, affinity chromatography, liquid chromatography,
electrophoresis, isoelectric focusing, salting-out, precipitation
with organic solvent, isoelectric point-precipitation, vacuum
concentration, hot air-drying and freeze-drying. The sequence of
two or more steps to be adopted for the isolation and purification
of the object substance may be arranged as desired and repetition
of certain treatment(s) may be done, if necessary.
According to one aspect of this invention, there is provided a
process for the preparation of a glycoprotein substance having an
average molecular weight of about 470,000 and possessing an
antitumor activity from scallop which comprises recovering the
liquid portion which comes from cooking of raw scallop with a hot
aqueous solvent or with vapor of such solvent and which is to be
discarded as waste, concentrating the liquid portion thus recovered
to a smaller volume or to dryness to yield a concentrate or dry
powder, dissolving the concentrate or dry powder in water or an
aqueous solvent to form an aqueous solution thereof and subjecting
the aqueous solution to a series of treatments comprising ion
exchange chromatography with a basic anion exchanger and molecular
weight-fractionating treatments (i.e. fractionating treatments for
isolating a fraction of aimed molecular weight range) comprising
gel-filtration and electrophoresis, in any desired sequence,
whereby isolating a fraction consisting essentially of a
glycoprotein substance which is adsorbable on a basic anion
exchanger and which has an average molecular weight of about
470,000.
A preferred embodiment of the process for the preparation of the
glycoprotein substance according to this invention comprises the
steps of recovering the liquid portion which comes from cooking of
raw scallop with a hot aqueous solvent or with vapor of such
solvent and which is to be discarded as waste, concentrating the
liquid portion thus recovered to a smaller volume or to dryness to
yield a concentrate or dry powder, dissolving the concentrate or
dry powder in water or an aqueous solvent to form an aqueous
solution thereof and subjecting the aqueous solution to ion
exchange chromatography with a basic anion exchanger to recover a
fraction which is adsorbed on the exchanger followed by subjecting
the fraction to molecular weightfractionating treatments comprising
gel-filtration and electrophoresis whereby to isolate a fraction
consisting essentially of a glycoprotein substance having an
average molecular weight of about 470,000. According to the most
preferred embodiment of the process according to this invention,
the treatments of the concentrate or dry powder are carried out in
a buffer solution having a strong buffering action.
The liquid portion coming from cooking of raw scallop to be used as
starting material according to the process of this invention may
include those by-produced when fresh or raw scallop which may have
been heated if desired, is cooked or heat-treated in a hot aqueous
solvent or with vapor of such solvent for the purpose of obtaining
edible portions thereof. The hot aqueous solvent to be used as
heating medium may include hot or boiling water, steam and other
hot aqueous solvents such as saline solution and sea water and
vapor of such solvents.
In order to take up edible portion of scallop and to recover the
liquid portion containing antitumor substances by cooking or
heat-treating (hereinafter referred to as "cooking"raw scallop in a
hot aqueous solvent or with vapor of such solvent, the cooking
operation is effected in one or more steps, in each of which the
scallop is brought into contact with a hot aqueous solvent in the
form of liquid and/or vapor which serves as both heating medium and
extracting solvent. The method of contact between scallop and hot
aqueous solvent may be selected as desired, for example from among
those of direct exposure to solvent vapor, direct pouring of hot
solvent and immersion into hot solvent. The hot aqueous solvent may
be used in the form of either liquid or vapor or both.
For the purpose of this invention, the raw scallop may be used in
its entirety, i.e. in the shell, or a shelled form with or without
liver, as desired. According to this invention, therefore, all the
edible portions, i.e. meat, ligament and the like, of scallop can
be used for food after the cooking operation because no cutting or
grinding of scallop is required for cooking.
Usually, the cooking may be carried out at a temperature of about
50.degree..about.120.degree. C., preferably about
60.degree..about.120.degree. C. for a period of time of about
3.about.120 minutes, preferably about 5.about.60 minutes.
The liquid portion coming from the cooking of raw scallop as
above-mentioned, which contains antitumor substances, may be
concentrated to a smaller volume or to dryness to yield a
concentrate or dry powder in a known manner, e.g. by heating in
vacuo, hot air-drying or freezedrying. Hot air-drying such as one
using a spray drier is preferred in order to prevent or minimize
the deterioration of the object substances because the contact
(residence) time is as short as about 5.about.80 seconds under
conditions of the hot air temperature of about
200.degree..about.350.degree. C. at the inlet and of about
80.degree..about.170.degree. C. at the outlet.
The isolation of the glycoprotein substance of this invention from
the concentrate or dry powder thus obtained and the subsequent
purification may be carried out, according to one embodiment of
this invention, as follows:
The concentrate or dry powder is dissolved in an aqueous solvent
and the solution is centrifuged or filtered to remove insoluble
matters and then desalted by gel-filtration or dialysis, for
example. The resulting solution is subjected to ion-exchange
chromatography by passing through a column of a basic anion
exchanger such as an ion-exchanging gel having such dissociating
group as diethylaminoethyl group or aminoethyl group with the
elution of the adsorbed fraction being effected with aqueous NaCl
solutions (about 0.07.about.0.4 mol/l) as eluents. The eluted
fractions are subjected to molecular weight-fractionating treatment
such as vertical slab gel-electrophoresis and gel-filtration to
obtain the object substance having an average molecular weight of
about 470,000. Optional step or steps for the purposes of desalting
and concentration may be inserted at any suitable point between
steps involved in the process, if desired. The desalting may be
effected typically by dialysis or gel-filtration and the
concentration may be carried out typically by heating in vacuo or
freeze-drying. In order to prevent the deposition of the object
substance into insoluble precipitate, these treatments should
preferably be carried out in a buffer solution having a strong
buffering action, for example a phosphate buffer solution having a
concentration of about 0.05 M or higher, preferably about 0.1 M or
higher. The term "a phosphate buffer solution" used herein means an
aqueous solution of a phosphate mixture of potassium
dihydrogenphosphate and disodium hydrogenphosphate.
The water-soluble glycoprotein substance according to this
invention is useful as antitumor agent with such particular
advantages that it has a wide antitumor spectrum without
appreciable cytotoxicity and that a noticeable tumor regression
effect can be achieved by adopting various administration routes
which are normally applied in the management of cancers. Thus,
intra-tumor, subcutaneous, intraperitoneal, intracutaneous,
intramuscular, or intravenous injections, if necessary, oral and
rectal administrations, or in external applications, coating,
instillation and other methods of administration are feasible.
The process of this invention is also advantageous in view of
environmental protection in that a substantial amount of organic
substances contained in the liquid portion coming from cooking of
raw scallop can be recovered as useful product with the result that
the organic contents of the waste liquor are substantially
reduced.
In the tests hereinafter given, the evaluation of antitumor
activity of the antitumor substances isolated was made by the
following method unless otherwise stated.
Sarcoma 180 tumor cells are maintained in ICR mice, 5.about.7 weeks
old, in an ascites form. Four millions of Sarcoma 180 cells are
subcutaneously transplanted into the right inguinal region of
female ICR mice, 6 weeks old. Then, the test samples dissolved or
suspended in a physiological saline in adequate concentrations
(injection volume, 0.1 ml) are subcutaneously injected into the
left inguinal region of the mice under test three times every
second day. Three weeks after the tumor cell transplantation, the
diameter of growing solid tumor or the weight of solid tumor
removed are measured and the data obtained are compared with those
of control group wherein a physiological saline solution is used in
place of the test sample solution.
The inhibition ratio and complete tumor regression are calculated
by the following formula: ##EQU1##
This invention is now illustrated with reference to the following
Examples to which the invention is in no way limited.
EXAMPLE 1
One part (by weight) of raw scallop shellfish (Patinopecten
yessoensis) in the shells was charged into a vessel continuously,
into which 0.10 parts (by weight) of superheated steam at
105.degree..about.110.degree. C. was blown so that the raw scallop
shellfish was directly exposed to the blown steam and cooked at
90.degree..about.100.degree. C. for 10 minutes. At the bottom of
the vessel, there was collected a volume of the water condensate
containing the active substances dissolved therein as a first crop
solution of the active substances. The outer shells of raw scallop
shellfish used as the starting material had about 0.1 parts (by
weight) of the infesting acorn shells attached thereto. The first
crop solution of the active substances was removed out of said
vessel and then slowly cooled down from 90.degree. C. to 50.degree.
C. and then immediately passed through a spraying drier. This drier
had an inlet through which a stream of hot air at 280.degree. C.
was passed into the drier, as well as an outlet through which the
effluent gases were discharged from the drier at a temperature of
125.degree. C. The first crop solution of the active substances was
dried in the drier in a retention time of 45 seconds to give a
first crude powder of the active substances (Sample 1a) in a yield
of 0.27% by weight based on the raw scallop employed.
Then, the ligament portions were removed from the scallop by means
of knife. The scallop ligament so collected (50 kg) was placed into
a volume (450 kg) of a boiling saline solution containing 10% by
weight of sodium chloride in water, boiled in the boiling saline
solution for 20 minutes and then removed therefrom. With the same
saline solution, further three 50 kg portions of the scallop
ligament were treated in the same manner as above. This saline
solution was recovered as a second crop solution of the active
substances. The second crop solution of the active substances was
slowly cooled down from 90.degree. C. to 40.degree. C. and dried in
the same manner as that for the first crop solution of the active
substances to give a second crude powder of the active substances
(Sample 1b) in a yield of 0.20% by weight based on the scallop
employed.
Two parts by weight of the first crude powder (Sample 1a) were
mixed with one part by weight of the second crude powder (Sample
1b) to give a third crude powder of the active substances (Sample
1c).
Test A
The third crude powder, i.e. Sample 1c, (1200 g) obtained as above
was dissolved in 6 l of a 0.01 M phosphate buffer solution (pH 7.5)
and the resulting solution was centrifuged at 10,000 G for 20
minutes to remove the insoluble matters therefrom. A 3 l portion of
the supernatant solution was charged for the desalting purpose into
a column (15 cm in height; 16 l in volume) of Sephadex G-25 (a
product of Pharmacia Fine Chemicals Co., which is a gel for
gel-filtration obtained by three-dimensionally crosslinking dextran
with epichlorohydrin and which is capable of fractionating
substances of molecular weights in the range of about
500.about.5000) which had been well equilibrated with distilled
water. Then, a further amount of distilled water was passed through
the column as developer and the eluate was continuously passed
through a conductometric titration cell to detect the point at
which the electric conductivity of the eluate was increased due to
the presence of inorganic salts therein, so that fractions eluted
by that point were collected. The fractions so collected were
passed through a column of an ion exchanging gel, DEAE-Sepharose
CL-6B (a product of Pharmacia Fine Chemicals, Co. which is a gel
obtained by three-dimensionally crosslinking agarose with
2,3-dibromopropanol followed by introducing diethylaminoethyl group
through an ether linkage and which has chlorine ion as counter ion,
the upper limit of molecular weights to be fractionated of about
1.times.10.sup.6 and the total exchange capacity of 15.+-.2 meq/100
ml) which had been well equilibrated with distilled water whereby
to remove non-adsorptive substances. The column was then eluted
with an eluent comprising a 0.01 M phosphate buffer solution (pH
7.5) containing 0.25 M NaCl. The eluted fractions containing
glycoprotein substances of different molecular weights were
collected and concentrated in vacuo below 30.degree. C. The
resulting concentrate was dialyzed against distilled water for 48
hours for desalting and then freeze-dried to yield 6.15 g of a
glycoprotein powder (A).
Another 3 l portion of the supernatant solution derived from Sample
1c was charged for the desalting purpose into a column of Sephadex
G-25 which had been equilibrated with a 0.1 M phosphate buffer
solution (pH 7.5), with the elution being effected by passing
through the column a further amount of the same 0.1 M phosphate
buffer solution as that used above as developer. The desalted
fractions were collected and passed through a column of
DEAE-Sepharose CL-6B which was the same as above, but which had
been equilibrated with the same 0.1 M phosphate buffer solution as
that used above whereby to remove non-adsorptive substances. The
column was then eluted with an eluent comprising a 0.1 M phosphate
buffer solution (pH 7.5) containing 0.2 M NaCl and the eluted
fractions were treated in the same manner as that used to treat the
corresponding eluted fractions derived from the first 3 l portion
above to yield 5.83 g of a glycoprotein powder (B).
The antitumor activity of each of powder (A) and powder (B) thus
obtained was examined according to the method defined above and the
results are shown in Table 1.
TABLE 1 ______________________________________ Dose Average
Inhibition (mg/mouse .times. weight of ratio Complete Sample
times*) tumors (g) (%) regression
______________________________________ Control -- 9.25 -- 0/8 A 10
.times. 3 1.15 87.6 0/5 B 10 .times. 3 1.20 87.0 0/5
______________________________________ *Administered into the tumor
site on the 5th, 7th and 9th days after the tumor cell
transplantation.
Test B
One gram each of powder (A) and powder (B) obtained in Test A above
was dissolved in 20 ml of a 0.1 M phosphate buffer solution (pH
7.5) and each solution prepared was charged into a column (60 cm in
height and 2.5 cm in inner diameter) of Sephacryl S-400 (a product
of Pharmacia Fine Chemicals Co. which is a gel obtained by
crosslinking allyldextran with N,N'-methylenebisacrylamide and
which is capable of fractionating substances of molecular weights
in the range of 1.times.10.sup.4 .about.2.times.10.sup.6) which had
been well equilibrated with the same 0.1 M phosphate buffer
solution as that used above. The column was eluted with the same
0.1 M phosphate buffer solution as that used above (pH 7.5) to
collect the eluate as fraction (A) for powder (A) and as fraction
(B) for powder (B), respectively.
Another 1 g portion of powder (B) was dissolved in 20 ml of a 0.01
M phosphate buffer solution (pH 7.5) and the solution was charged
into a column of Sephacryl S-400 which was similar to that used
above but which had been well equilibrated with distilled water.
The column was eluted with distilled water whereby to collect the
eluate as fraction (C).
In order to decide the ranges of molecular weights to be
fractionated from the three fractions (A), (B) and (C), a
calibration curve was depicted from elution data of known standard
substances such as blue dextran, thyroglobulin, ferritin, aldolase,
ovalbumin and ribonuclease A on the column of Sephacryl S-400 same
as above. Thus, the three molecular weight fractions, namely,
fraction (1) with molecular weights higher than 300,000, fraction
(2) with molecular weights of 100,000.about.300,000 and fraction
(3) with molecular weights lower than 100,000, were decided
relatively using the calibration curve.
The three fractions (A), (B) and (C) each were further fractionated
into the fractions (1), (2) and (3), respectively and the
respective fractions were dialyzed against distilled water followed
by freeze-drying to yield nine powders A-1, A-2, A-3, B-1, B-2,
B-3, C-1, C-2 and C-3. The yield of each powder obtained is shown
in Table 2.
TABLE 2 ______________________________________ Fraction Fraction
Fraction Fraction used Developer 1 2 3 Total
______________________________________ A Buffer A-1: A-2: A-3: 0.75
g solution 0.11 g 0.42 g 0.22 g B Buffer B-1: B-2: B-3: 0.92 g
solution 0.20 g 0.47 g 0.24 g C Distilled C-1: C-2: C-3: 0.81 g
water 0.09 g 0.45 g 0.26 g
______________________________________
The results shown in Table 2 showed a considerable difference in
the yield of fraction 1 having molecular weights higher than
300,000 among A, B and C series experiments which were carried out
by using the same starting material and substantially thc same
method except for the use of different developers. Therefore, the
evaluation of antitumor activity was made on all the nine powder
fractions according to the method defined as above. The results are
shown in Table 3.
TABLE 3 ______________________________________ Dose Average
Inhibition Sample (mg/mouse .times. weight of ratio Complete tested
times*) tumors (g) (%) regression
______________________________________ Control -- 10.43 -- 0/8 A-1
5 .times. 3 5.83 44.1 0/5 A-2 " 2.73 73.8 0/4 A-3 " 2.68 74.3 0/5
B-1 " 1.95 81.3 1/5 B-2 " 2.43 76.7 0/4 B-3 " 2.15 79.3 0/4 C-1 "
6.28 39.8 0/4 C-2 " 2.80 73.2 0/5 C-3 " 2.53 75.7 0/4
______________________________________ *Administered into the tumor
site on the 5th, 7th and 9th days after the tumor cell
transplantation.
There was observed no appreciable difference in antitumor activity
between powders A and B in Test A. In contrast, comparison among
the highest molecular weight powder fractions A-1, B-1 and C-1 in
Test B clearly showed that fraction B-1 contained a substance
exhibiting the highest antitumor activity, whereas fraction C-1
contained little or no such substance of the highest antitumor
activity which must have been contained originally in powder B.
This suggested that the method used to recover fraction C from
powder B was not appropriate for the recovery of glycoprotein
substances having molecular weights higher than 300,000 possibly
due to their conversion or inclusion into insoluble materials
during the treatments or for some other reason.
Since there was nothing reported or even suggested so far about
that fractions of molecular weights higher than 300,000 derived
from shellfish contain substance or substances possessing antitumor
activities, we did not expect to be able to isolate such useful
substance(s) from the concentrate or dry powder derived from
scallop, but now found as above that such high molecular weight
substances which are difficult to keep in solution during
chromatographic treatments could be successfully treated by
ion-exchange chromatography, gel-filtration chromatography and the
like if eluent or developer to be used for said treatments is of a
strong pH buffering action. Based on these findings, we further
continued our study on such fractions of molecular weights higher
than 300,000 and finally ascertained the entity of substance
exhibiting the highest antitumor activity contained in such high
molecular weight fractions to be a glycoprotein substance which
shows a single band in 7.5% polyacrylamide gel-electrophoresis and
which has an average molecular weight of about 470,000.
EXAMPLE 2
(a) The third crude powder, i.e. Sample 1c, (600 g) obtained in
Example 1 was dissolved in 3 l of a 0.1 M phosphate buffer solution
(pH 7.5) and the resulting solution was centrifuged at 10,000 G for
20 minutes to remove insoluble matters therefrom and then charged
for the desalting purpose into a column (15 cm in height; 16 l in
volume) of Sephadex G 25 which had been well equilibrated with the
same 0.1 M phosphate buffer solution as that used above. Then, a
further amount (20 l/hr) of the same 0.1 M phosphate buffer
solution was passed through the column as developer and the eluted
fractions free from inorganic salts were collected by monitoring
the electric conductivity of the eluent in the same manner as that
used in Test A of Example 1. The fractions thus desalted were then
passed through a column of 16 l of a DEAE-Sepharose CL-6B
ion-exchanging gel which had been well equilibrated with the same
0.1 M phosphate buffer solution as that used above whereby to
remove non-adsorptive substances. The column was then washed well
with the same 0.1 M phosphate buffer solution as that used above
and eluted with an eluent comprising the same 0.1 M phosphate
buffer solution (pH 7.5) as that used above but containing 0.2 M
NaCl. The eluted fractions were collected and concentrated in vacuo
below 30.degree. C. to a volume (2 l) of about one-fifth of the
original volume. The concentrated eluate was dialyzed against
distilled water for 48 hours for desalting and then freeze-dried to
yield 6.08 g of a glycoprotein powder (C) (yield: 1.01% based on
the liquid portion coming from cooking of raw scallop).
A 2 g portion of powder (C) thus obtained was dissolved in 30 ml of
the same 0.1 M phosphate buffer solution (pH 7.5) as that used
above and the resulting solution was charged into a column (2.5 cm
in inner diameter and 60 cm in length) of Sephacryl S-400 which had
been well equilibrated with the same 0.1 M phosphate buffer
solution as that used above and chromatographed with the same 0.1 M
phosphate buffer solution as that used above as eluent. Fractions
corresponding to those comprising substances having molecular
weights higher than 300,000 which were relatively estimated by a
calibration curve depicted from elution data of known standard
substances on the same Sephacryl S-400 column as above were
collected and the fractions so collected were dialyzed against
distilled water for 48 hours followed by freeze-drying to yield 275
mg of powder (D). The elution pattern depicted in this
gel-filtration chromatography is given in FIG. 1.
A 20 mg portion of powder (D) was dissolved in 1.5 ml of a 0.1 M
phosphate buffer solution (pH 8.0) and the resulting solution was
subjected to vertical slab gel-electrophoresis using 7.5%
polyacrylamide gel in the same 0.1 M phosphate buffer solution as
that used above under the condition of constant current of 50 mA
for 4 hours whereby to fractionate substances according to
molecular weights. "Polyacrylamide gel-electrophoresis" is
electrophoresis using a gel bed (14 cm.times.14 cm.times.2 mm)
prepared by polymerizing a liquid mixture comprising acrylamide
monomer, N,N'-methylenebisacrylamide crosslinking agent and a
polymerization catalyst. "7.5% polyacrylamide" in the
electrophoresis means the concentration of acrylamide used in the
liquid mixture to be polymerized being 7.5% by weight. This
electrophoresis operation was repeated for two further 20 mg
portions of powder (D). In each of three operations, the portion of
gel which showed Rf values of 0.about.0.08 relatively to
Bromophenol blue at pH 8.0 was cut off and all three portions were
finely divided in 100 ml of an eluent comprising a 0.1 M phosphate
buffer solution (pH 7.5) containing 1 M NaCl on a homogenizer and
filtered. The gel separated on the filter was washed with further
amount of the eluent and the washings were combined with the
filtrate and the combined liquor was concentrated, freeze-dried,
dialyzed for 48 hours and finally freeze-dried to yield 12 mg of
powder (E).
(b) Powders (D) and (E) obtained in Example 2(a) above were
evaluated on their antitumor activities according to the test
method defined hereinbefore with the following results:
TABLE 4 ______________________________________ Dose Average
Inhibition (mg/mouse .times. weight of ratio Complete Sample
times*) tumors (g) (%) regression
______________________________________ Control -- 8.95 -- 0/9 D 5
.times. 3 1.48 83.4 1/4 1 .times. 3 1.92 78.5 0/5 E 1 .times. 3
0.53 94.1 2/5 0.5 .times. 3 0.42 95.3 1/4 0.2 .times. 3 0.45 94.9
2/5 ______________________________________ *Administered into the
tumor site on the 5th, 7th and 9th days after the tumor cell
transplantation.
(c) The average molecular weight of powder (E) obtained in Example
2(a) above was measured by SDS-5% polyacrylamide
gel-electrophoresis relatively to molecular weightreference
standard materials such as blue dextran, thyroglobulin, ferritin,
aldolase, ovalbumin and ribonuclease A in a usual manner.
"SDS-polyacrylamide gel-electrophoresis" is electrophoresis using a
gel prepared by polymerizing a liquid mixture comprising acrylamide
monomer, N,N'-methylenebisacrylamide crosslinking agent, a
polymerization catalyst and sodium dodecyl sulfate (SDS). The
electrophoresis of powder (E) showed a single band with color
development at the position corresponding to about 470,000
(accuracy: .+-.30,000).
(d) The isoelectric point of powder (E) obtained in Example 2(a)
was measured by isoelectric focusing over pH range of 4.0.about.6.0
under the condition of constant power of 20 W using Servalyt
Precotes 4-6 (a product of Serva AG which is a gel comprising
Servalyt AG 4-6 and 5% polyacrylamide gel) as precoated film for
isoelectric focusing. Servalyt AG 4-6 is a product of Serva AG,
which is a mixture of amphoteric electrolytes comprising aliphatic
compounds having molecular weights ranging 500.about.800 and
containing secondary and tertiary nitrogen-containing groups such
as guanid group, sulfonic acid group and phosphonic acid group and
is used as carrier ampholytes to give a pH gradient of
4.0.about.6.0. The isoelectric point of powder (E) was measured
relatively to those of known substances as reference standards by
comparing their migration bands and estimated to be about pH
5.5.
(e) Physico-chemical properties of powder (E) were also measured,
which are as shown hereinbefore.
EXAMPLE 3
(a) Antitumor activity of powder (E) obtained in Example 2, when
administered not only into the tumor site but also through other
routes, was examined by the following method.
Sarcoma 180 tumor cells (about 6.times.10.sup.6 cells) were
subcutaneously transplanted into the right groin of female ICR
mice, 6 weeks old. Powder (E) was dissolved in a physiological
saline in appropriate concentrations and each of the resulting test
solutions (pH 7.0; injection volume 0.1 ml) was injected on 5th,
7th and 9th days after the tumor cell transplantation. The
injections were effected through several routes, namely directly
into the tumor site, intravenously into the tail, intraperitoneally
and subcutaneously into the opposite groin. After the lapse of 5
weeks from the transplantation, the mice were killed and the solid
tumors were dissected out and weighed. The results were compared
with those of control group wherein a physiological saline solution
was administered in place of each test sample. Inhibition ratio (%)
and complete regression were calculated as defined hereinbefore.
The results are shown in Table 5.
TABLE 5 ______________________________________ Dose Average
Inhibition Administration (mg/mouse weight of ratio Complete route
.times. times) tumors (g) (%) regression
______________________________________ Control -- 9.23 -- 0/7
Directly into 0.1 .times. 3 0.45 95.1 1/5 tumor site Intravenously
0.2 .times. 3 0.32 96.5 3/4 into tail 0.1 .times. 3 1.52 83.5 0/4
Subcutaneously 0.2 .times. 3 0.43 95.3 1/5 into groin 0.1 .times. 3
0.51 94.4 2/5 Intraperito- 0.2 .times. 3 0 100 3/3 neally 0.1
.times. 3 1.11 88.0 1/4 ______________________________________
The results given in Table 5 demonstrate that powder (E) isolated
in Example 2 is remarkably effective in degeneration and complete
regression of Sarcoma 180 solid tumors through various routes for
administration, that is in tumor site, intravenously,
intraperitoneally and subcutaneously.
(b) Direct cytotoxicity of powder (E) was also examined against L
5178 Y Lymphoma cells in vitro, but there was found no appreciable
cytotoxicity.
The test on cytotoxicity was effected as follows:
L 5178 Y (5.times.10.sup.5 cells/ml) were suspended in a tissue
culture medium, Eagle MEM (containing 15% calf serum) and the
suspension, after the addition of test sample in the concentrations
as undermentioned thereto, was incubated in an incubator under 5%
CO.sub.2 in air at 37.degree. C. for 48 hours. Then, the effect of
test sample on target cell proliferation was estimated with a
phase-contrast microscope. A physiological saline solution and
mitomycin C were used as control and reference, respectively. The
results of the direct cytotoxicity test are shown in Table 6.
TABLE 6 ______________________________________ Concentration Growth
of sample inhibition Sample (.mu.g/ml) (%)
______________________________________ Powder (E) of 40 0 this
invention 200 0 Mitomycin C 20 30 100 68 Control 0 (Physiological
saline solution) ______________________________________
EXAMPLE 4
This Example illustrates the antitumor activity of powder (E)
isolated in Example 2 on several solid tumors other than Sarcoma
180 solid tumor.
The tumor cells (4.times.10.sup.6 cells) of each of Ehrlich
carcinoma, Leukemia SN-36, NTF reticulum cell sarcoma and
methylcholanthrene-induced Fibrosarcoma which were maintained in
ICR female mice or Balb/c mice (for Fibrosarcoma only) were
subcutaneously transplanted into one groin of another group of
female, 6 weeks aged ICR mice or Balb/c mice correspondingly. One
week after the transplantation, the tumor cells were confirmed to
have grown to solid tumors. Then, a solution in physiological
saline of powder (E) was directly injected into the tumor site at a
dose of 0.2 mg/mouse three times on alternate days. Five weeks
after the tumor cell transplantation, the solid tumors were
dissected out and weighed. The results were compared with that of
control group wherein a physiological saline solution was
administered in place of the test solution. Inhibition ratio (%)
and complete regression were calculated as hereinbefore defined.
The results are shown in Table 7. 7.
TABLE 7 ______________________________________ Ehrlich carcinoma
SN-36 NTF Fibrosarcoma ______________________________________
Inhibition 100 83.9 93.8 100 ratio (%) Complete 4/4 2/4 3/4 4/4
regression ______________________________________
EXAMPLE 5
The procedure described in Example 2 was repeated except that
Sample 1a was used in place of Sample 1c, yielding an antitumor
substance consisting essentially of a water-soluble glycoprotein
substance having an average molecular weight of about 470,000. The
physico-chemical properties and antitumor activity of this
substance were substantially the same as those of powder (E)
isolated in Example 2.
EXAMPLE 6
The procedure described in Example 2 was repeated except that
Sample 1b was used in place of Sample 1c, yielding an antitumor
substance consisting essentially of a water-soluble glycoprotein
substance having an average molecular weight of about 470,000. No
difference could be found between the physico-chemical properties
and antitumor activity of this substance and those of powder (E)
isolated in Example 2.
* * * * *